This application relates to a method for the detection of mutations, including previously unknown mutations, in a gene or a gene fragment having a known wild-type sequence.
Many diseases and conditions have been found to be associated with genetic mutations, and more such associations are being identified as time goes by. In some cases, such as sickle-cell anemia, a single base change has been identified as the causative mutation. More generally, however, many different mutations may manifest themselves as a single disease. To test for each of these mutations individually using hybridization-based tests would require the development of as many different probes as there are mutations. Such an effort would involve substantial expense, however, such that in most cases hybridization-based diagnostics are only available for the most prevalent mutations. Furthermore, because development of mutation-specific hybridization-based diagnostics requires knowledge of the mutant sequence, development of diagnostic tests must await the identification and characterization of any given mutation in at least one individual.
The present invention overcomes some of the limitations of hybridization-based diagnostics by providing an assay which relies only upon knowledge of the wild-type sequence, and which detects all types of mutations, i.e., point mutations, insertions and deletions. The method involves the use of a set of oligonucleotide probes which hybridize in series along the length of the gene; the ligation of the probes together to form ligation products; and the evaluation of the sizes and quantities of the ligation products. When the gene being analyzed corresponds to the normal sequence, a characteristic pattern of ligation products is formed, including a substantial amount of full length product which results from all of the probes in the set being ligated together. When a mutation appears in the gene, the hybridization of the probe overlapping the mutation is impaired, with the result that some or all of the ligation product is of smaller size. By evaluating the sizes and quantitites of the ligation products; both the existence of a mutation and its approximate position can be identified. A definitive identification of the mutation can then be made by direct sequencing over a very restricted set of positions.
EP-A-0 185 494 discloses an assay for detection of specific nucleotide sequences in which two oligonucleotide probes which perfectly match adjacent portions of the expected sequence are hybridized and then ligated together. The assay is said to improve reliability over a plain hybridization assay, since it is unlikely that both probes would bind to a spurious location in ligatable proximity.
Ligase enzymes have previously been used in diagnostics for the detection of known point mutations. As described in Landegren et al., "A Ligase-Mediated Gene Detection Technique", Science 241: 1077-1080 (1988), and as shown in FIG. 1, this technique involved hybridization of two probes 1 and 1' to the target gene fragment 2 being analyzed. The probes 1 and 1' are selected such that the terminal base in one fragment aligns with the known mutation site when this probe is hybridized with the target gene fragment. The terminal base of the other probe aligns with the base immediately adjacent to the known mutation site. When the probes are completely complementary to the target sequence, T4 DNA ligase will couple the two probes together into a single fragment. When a mutation is present, however, the absence of hybridization at the end of one of the probes will prevent (or at least substantially reduce) the amount of ligation which occurs. By labeling one probe fragment with a capture moiety such as biotin, and the other probe fragment with a radiolabel, Landegren et al. were able to identify the presence or absence of the specific point mutation based upon formation (or non-formation) of the ligated molecule which incorporated both the capture moiety and the radiolabel.
The basic technique described by Landegren et al. has been modified by a number of researchers. For example, F. Barany, "Genetic Disease Detection and DNA Amplification Using Cloned Thermostable Ligase", Proc. Nat'l Acad. Sci. USA 88: 189-193 (1991) described the use of a thermostable ligase in place of the T4 DNA ligase utilized by Landegren et al. This enzyme allowed the use of higher temperatures which improved the discrimination between perfectly matched and mismatched sequences. Like the basic Landegren et al. technique, however, Barany only addressed detection of the known point mutations using a pair of probes which meet at the site of the mutation.
A further modification of the Landegren et al. technique is described in PCT Patent Publication No. WO 94/08047 entitled "Ligase Chain Reaction Method for Detection of Small Mutations." In this application, deletion mutations of up to about 5 bases are detected using ligation of a pair of probes. As in the basic Landegren technology, however, a complete knowledge of the site and nature of the mutation is necessary to carry out the test.
It is an object of the present invention to overcome this fundamental limitation of known tests which utilize ligase, and to provide a test methodology which permits the detection of mutations including previously unknown and uncharacterized mutations, whether large or small in magnitude, within a gene of clinical or diagnostic importance.
It is a further object of the present invention to provide kits which may be used in carrying out this method.